POS0256 IDENTIFICATION OF TWO BIOLOGICAL SUBTYPES OF CRPS
Authors: M. Perez Vertti Valdes, A. Juengel, I. Kolm, L. Opitz, H. Rehrauer, O. Distler, F. Brunner, S. Dudli
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Patients with Complex Regional Pain Syndrome (CRPS) present prolonged and debilitating pain and tissue damage. To date, treatment remains a challenge. Biological phenotyping has not been performed yet, and thus, molecular therapies are missing. <h3>Objectives</h3> To determine whether different biological CRPS subtypes exist and to provide a molecular profile of these subtypes. <h3>Methods</h3> CRPS patients fulfilling the <i>Budapest criteria</i> were included. Body Perception Disturbance (BPD) and pain scores (0-100) questionnaires were recorded. Skin punch biopsies (3 millimetres) from the affected and non-affected sides (n=6 + 6) and matching blood samples were taken. Biopsies were split in two for transcriptomic and histological analyses. Blood was processed to collect plasma after centrifugation and peripheral blood mononuclear cells (PBMCs) with a Ficoll gradient. Total RNA was isolated from skin biopsies and from PBMCs for bulk sequencing. Differentially expressed genes (DEGs) were determined using DESeq2 generalised linear model (p≤0.01, log2 ratio≥0.5). Hierarchical clustering and principal component analysis (PCA) were performed on raw transcriptomic data to identify patient sub-populations. Cell types were approximated in skin and PBMCs with deconvolution analysis using RNA sequencing databases with known cell frequencies (PRESS cohort and immunedeconv, respectively). Twenty pro- and anti-inflammatory plasma cytokines were measured using chemiluminescence-based assays (MSD U-PLEX Viral Combo 1). Histological slides were scored (0-3) blinded by a dermato-pathologist based on a panel of 10 morphological and cellular characteristics. Protein levels, histological evaluations and demographic data were compared between CRPS clusters using unpaired t-tests. <h3>Results</h3> Six women with CRPS 1 of the hand (n=5) and foot (n=1) participated in the study. Mean age was 47.3 ± 14.2 yrs and mean symptom duration was 57.8 ± 44.2 weeks. Skin transcriptomics showed 427 DEGs when comparing affected vs. non-affected samples (<b>Figure 1a</b>). Samples clustered by patient rather than affected/non-affected side (<b>Figure 1b</b>). PCA of only the affected samples displayed two clusters (PC1=56%, PC2=17%) (<b>Figure 1c</b>). Further evaluation of all affected samples by deconvolution analysis suggested two distinct cellular signatures, clustering patients in the same way as the PCA. Keratinocyte-related genes were upregulated in cluster 1 (n=3) and strong fibroblast, microvascular endothelial cell, monocyte and M1 macrophage signatures were found in cluster 2 (n=3) (<b>Figure 1d</b>). Consequently, gene ontology (GO) of each cluster showed enrichment of epidermis development and downregulation of peptidase terms in cluster 1 and extracellular matrix and collagen fibril organisation in cluster 2. Histopathological examinations did not show any difference between subtypes. PCA of PBMCs transcriptome validated the same distribution of patients into two clusters (PC1=62%, PC2=16%) (<b>Figure 1e</b>). A total of 2'328 DEGs were found between both clusters, with an overrepresentation of immune and inflammatory response, and neutrophil degranulation and chemotaxis-related terms in cluster 1 (n=2) according to GO. Deconvolution analysis confirmed the presence of a stronger signature of neutrophils and M2 macrophages in cluster 1 than cluster 2. Blood cytokine quantification showed that interleukin-1 receptor antagonist (IL-1RA) levels were 1.9 times higher in cluster 1 than in cluster 2 (n=3 + 3, p=0.02) (<b>Figure 1f</b>). Consistently, transcriptomic data indicated that cluster 1 had higher expression of IL-1RA encoding gene (IL-1RN) in matching skin (log2 ratio=0.88, FDR=0.06) and PBMCs (log2 ratio=1.1, FDR≤0.01,) than cluster 2. There was no significant difference in age or symptoms duration between clusters, but pain and BPD questionnaires revealed differences between groups, suggesting that the clusters might be clinically distinguishable (<b>Figure 1g</b>). <h3>Conclusion</h3> Our findings suggest the existence of two biological CRPS subtypes. Molecular patterns could facilitate the development of targeted therapies. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Melina Perez Vertti Valdes: None declared, Astrid Juengel: None declared, Isabel Kolm: None declared, Lennart Opitz: None declared, Hubert Rehrauer: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant Siences, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Pfizer, Prometheus, Redxpharna, Roivant, Sanofi and Topadur, Grant/research support from: BI, Kymera, Mitsubishi Tanabe, Florian Brunner: None declared, Stefan Dudli: None declared.